PROJECT SUMMARY Engineering gold nanoparticles (GNPs) are very attractive for many biomedical applications and offer high clinical potential for advanced diagnosis and therapy. However, despite favorable profile of GNPs, the growing number of recent reports have indicated their undesired effects. Specifically, when GNPs disseminate through the organism by blood vessels (e.g., after commonly used intravenous injection) they interact with endothelial cells lining the inner surface of blood vessels and may cause vascular dysfunctions and cardiovascular disorders. Simultaneously with blood dissemination, GNPs commonly and easily penetrate lymphatic vessels and directly contact with lymphatic endothelial cells (LECs). The impact of GNPs on LECs and lymphatic vessels is unknown. The goal of this proposal is to study the interaction between GNPs and lymphatic vessels by monitoring of GNP effects on single LECs and individual isolated lymphatic vessels in vitro, and, then, on lymphatic function in vivo. Our central hypothesis is that GNPs cause dose- and time-dependent dysfunction of lymphatic vessels through direct interaction with LECs. We suggest that molecular mechanisms of GNP-induced LEC dysfunction involve activation of endothelial nitric oxide synthases, increasing production of nitric oxide and reactive oxygen species which inhibit contractile activity and lymph flow in lymphatic vessels. To accomplish this project we plan to integrate our expertise in lymphatic research, single-cell analysis and nanomedicine with the power of technological platforms that were developed by our team, including photoacoustic flow cytometry and high-resolution photothermal microscopy. We will pursue our goal through the following specific aims: (1) Explore molecular mechanisms of GNP-LEC interaction; (2) Determine whether GNPs induce dysfunction of isolated LV through interaction with LECs; (3) Define the impact of GNPs on LV function in vivo. Successful completion of these aims will advance our understanding of the interaction between GNPs and lymphatic vessels. Thus, for the first time, we will answer the critical and clinically relevant question of whether GNPs can induce dysfunction of lymphatic vessels, and whether this dysfunction is a result of interaction between GNPs and lymphatic endothelium. Overall, in the context of its impact, the knowledge gained from this research will help to select safe GNPs for clinical translation and addresses the needs of the broader nanotechnology community to develop comprehensive biological response profiles for an engineering nanomaterials that is in line with the Strategic Plan of National Nanotechnology Initiative for 2014-2015.